Human-Cell Mutagens in Respirable Airborne Particles in the

The mutagenic potency (induced mutant fraction per μg organic carbon) of the ... One method that has been widely used is mutagenicity testing (6−13...
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Environ. Sci. Technol. 2004, 38, 682-689

Human-Cell Mutagens in Respirable Airborne Particles in the Northeastern United States. 1. Mutagenicity of Fractionated Samples D A N I E L U . P E D E R S E N , †,‡ J O H N L . D U R A N T , * ,§ BRUCE W. PENMAN,+ CHARLES L. CRESPI,| HAROLD F. HEMOND,† ARTHUR L. LAFLEUR,⊥ AND GLEN R. CASS+ Department of Civil & Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, Department of Civil & Environmental Engineering, Tufts University, Medford, Massachusetts 02155, Gentest Corporation, 6 Henshaw Street, Woburn, Massachusetts 01801, and Center for Environmental Health Sciences, Massachusetts Instiute of Technology, Cambridge, Massachusetts 02139

Few studies have characterized the regional scale (300500 km) variability of the mutagenicity of respirable airborne particles (PM2.5). We previously collected 24-h PM2.5 samples for 1 year from background, suburban, and urban sites in Massachusetts (MA) and rural and urban sites in upstate New York (NY) (n ) 53-60 samples per site). Bimonthly composites of these samples were mutagenic to human cells. The present report describes our effort to identify chemical classes responsible for the mutagenicity of the samples, to quantify spatial differences in mutagenicity, and to compare the mutagenicity of samples composited in different ways. Organic extracts and HPLC fractions (two nonpolar, one semipolar, and one polar) of annual composites were tested for mutagenicity in the h1A1v2 cells, a line of human B-lymphoblastoid cells that express cytochrome P450 CYP1A1 cDNA. The mutagenic potency (induced mutant fraction per µg organic carbon) of the semipolar fractions was the highest at all five sites, accounting for 35-82% of total mutagenic potency of the samples, vs the nonpolar (4-38%) and polar (14-32%) fractions. These results are consistent with previous studies. While unfractionated extracts exhibited no spatial variations, the mutagenicity of semipolar fractions at the NY sites was ∼2-fold higher than at the MA sites. This suggests there may be significant regional differences in the sources and/ or transport and transformation of mutagenic compounds * Corresponding author phone: 617-627-5489; fax: 617-627-3994; e-mail: [email protected]. † Department of Civil & Environmental Engineering, Massachusetts Institute of Technology. ‡ Current address: Air Quality Laboratory, Institute of Earth Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. § Tufts University. | Gentest Corp. ⊥ Center for Environmental Health Sciences, Massachusetts Institute of Technology. + Deceased. 682

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in PM2.5. In addition, mutagenic potency was sensitive to whether samples were fractionated and how they were composited: unfractionated annual composite samples at the NY sites were significantly less mutagenic than their semipolar fractions and the annual average of bimonthly composites; spatial differences in the mutagenic potency of bimonthly composites and the semipolar fractions were not apparent in the annual composites.

Introduction Airborne particles, whether natural or anthropogenic, are typically composed of complex mixtures of organic and inorganic compounds. Exposure to airborne particles has been linked to lung cancer and other cardiopulmonary diseases (1-5), and it has been hypothesized that specific chemical constituents of the particles may be causative. A variety of methods have been employed to measure the toxicity of airborne particles. One method that has been widely used is mutagenicity testing (6-13). Mutagenicity, the ability of physical and chemical agents to cause inheritable changes in DNA, is used as a screening tool for relative genotoxicity, providing a basis for selecting samples for more in-depth analysis including chemical characterization. In addition, variations in mutagenicity may offer evidence about the sources, transport, and transformations of airborne particles. Previously we described seasonal and spatial variations in the human-cell mutagenicity of bimonthly composites of PM2.5 collected in upstate New York (NY) and Massachusetts (MA), in the northeastern United States (14). We observed that the mutagenic potency (induced mutant fraction per µg of organic carbon) was ∼2-fold higher in winter months than in summer months, and the annual average of mutagenic potency for NY was ∼2-fold higher than in MA. Mutagen density (induced mutant fraction per m3 of air) was ∼2-fold higher in urban centers than in nearby rural areas. The goal of the current study was to determine if there were significant differences in the major classes of compounds responsible for the human-cell mutagenicity in the samples collected previously in NY and MA. Our strategy was to (1) fractionate the samples into distinct chemical classes, (2) test the fractions for mutagenicity and identify fractions that were significantly mutagenic, and (3) assess local and regional differences in the mutagenicity of the sample extracts and their fractions. A fourth step was to compare the mutagenicity of fractionated annual composite samples against that of bimonthly composites and separate wintertime samples to determine if our interpretation of mutagenicity trends was sensitive to the methods used to composite samples. To our knowledge, this is the first regional comparison of the mutagenicity of fractionated PM2.5 samples, especially across a wide range of urbanization.

Experimental Methods Details of the methods used for sample collection, preparation, and mutagenicity testing have been described previously (14) and are summarized here. Sampling Locations and Methods. Sampling was conducted at five sites located between Lake Ontario and the Atlantic Ocean, representing a wide range of land use. Sites were established in Kenmore Square (KS) in downtown Boston and in Reading (RD), a residential community 20 km north of Boston. A regional background site was established at Quabbin State Park (QB), a protected watershed in central 10.1021/es0347282 CCC: $27.50

 2004 American Chemical Society Published on Web 12/12/2003

FIGURE 1. HPLC fractionation program and representative polycyclic aromatic compounds that elute in each fraction. The fractions are nonpolar 1 (NP1, cutoff time 350 s), nonpolar 2 (NP2, 600 s), semipolar (SP, 2650 s), and polar (P, 3800 s). Solvents are hexane, dichloromethane (DCM), and isopropyl alcohol (IPA). MA, 120 km west of KS. Two inland sites were located in Rochester (RO), NY, an urban center 550 km west of KS, and Brockport (BR), NY, a rural location 25 km west of RO. Airborne particles larger than ∼3 µm were collected for 24-h every sixth day of 1995, simultaneously at all sites, on quartzfiber filters using a ∼300 L/min dichotomous virtual impactor (15, 16). Filters were prebaked at 550 °C to minimize their organic carbon content, collected the day after sampling, and stored at -20 °C. Samples were also collected on 20 days between November 29 and December 20, 1994, at RD and KS, yielding two composite samples representative of wintertime ambient air in eastern MA. Sample Preparation. Annual composite samples were prepared by cutting 1/6 portions of each of the filters collected at a given site (number of samples: BR n ) 60, RO n ) 59, RD n ) 57, QB n ) 56, KS n ) 53), which were then Soxhletextracted together in 100 mL of HPLC-grade dichloromethane (CH2Cl2) for 24 h. The extraction was repeated for an additional 24 h in 100 mL of fresh CH2Cl2 to capture slowly desorbing organic compounds. Extracts were concentrated (not to dryness) under a gentle stream of N2 and then filtered through a 0.2 µm Teflon filter. Organic and elemental carbon (OC and EC) content on the filters was measured by a thermal evolution and combustion method (17). The OC mass measured on a filter prior to extraction is defined as equivalent organic carbon (EOC). The CH2Cl2 extraction has been found to yield ∼93% of the EOC (12). Mutagenicity results are presented per µg of EOC on the original filters. Portions of the extracts were fractionated by normal-phase HPLC. A Beckman Gold HPLC system with a preparatory cyanopropyl (CN) column (250 mm length × 10 mm i.d.) operating at 4 mL/min was used to separate compounds into two nonpolar fractions (NP1 and NP2), a semipolar

fraction (SP), and a polar fraction (P). CN has been shown to preserve the mutagenicity of complex mixtures during fractionation (18). The solvent program began at 95:5 hexane: CH2Cl2 for 20 min, ramped to 100% CH2Cl2 over 10 min and held for 10 min, ramped to 50:50 CH2Cl2:isopropyl alcohol over 10 min and held for 20 min (Figure 1). Cutoff times and selected compounds that elute in each of the four fractions are indicated in Figure 1. The cutoff times (350, 600, 2650, 3800 s) were chosen to isolate different groups of humancell mutagens in each fraction. The majority of lower molecular weight (MW) PAHs are contained in the NP1 fraction, along with most of the alkanes and other nonaromatic organics, which are not considered to be mutagenic (19). The NP2 fraction contains PAH of MW between 176 and 276 amu, which include such human-cell mutagens as benzo[a]pyrene, while the SP fraction contains PAH of MW 276 amu and greater as well as semipolar PAC such as ketones, quinones, and nitro-PAH, some of which are mutagenic in human-cell assays (20, 21). The P fraction contains polar PACs, such as carboxylic acids, dicarboxylic acids, and hydroxy PACs. Recovery of polycyclic aromatic hydrocarbons (PAH) using this fractionation method was reported to be 77-110% (22). To determine whether mutagenicity was lost during fractionation, reconstituted extracts (R) were assembled for RO and KS by combining equal portions of all four fractions. Human-Cell Mutation Assay. Immediately prior to mutagenicity testing, aliquots from each extract and its fractions were exchanged into dimethyl sulfoxide (DMSO). The mutagenicity of the samples was measured in the h1A1v2 line of human B-lymphoblastoid cells, which constitutively overexpresses human cytochrome P450 1A1 (CYP1A1), an enzyme involved in the metabolism of many promutagens VOL. 38, NO. 3, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Dose/response plots for annual composite extracts and fractions. Units on the vertical axis are induced mutant fraction per million [IMF×106]. Multiple points at a given dose represent independent assays. Error bars represent 1 SD of the experimental mean. The bold lines are linear least-squares fits forced through the origin. The mutagenic potency is equal to the slope of this line ((error of the fit) in units of IMF×106 per µg equivalent organic carbon (EOC), given with r2 values. (1) Indicates that results are sensitive to assumption of linearity (see Experimental Section). (22-24). Each extract was tested at three doses in duplicate 12 mL cell cultures in at least two independent assays with four negative controls (60 µL DMSO) and two positive controls (1.0 µg mL-1 benzo[a]pyrene). Point mutations and loss of heterozygocity were measured at the thymidine kinase (tk) locus after 72 h. The mutant fraction (25) induced in excess of the negative controls is presented in units of induced mutant fraction per million (IMF×106). Two measures were used to quantify the mutagenicity of the extracts. Mutagenic potency (IMF×106 per µg of EOC) is the slope of the linear regression fitted to the concentration-response plot. A comparison of different regression conditions (e.g., whether to force the fit through the origin or to subtract concurrent negative controls) demonstrated that the results were insensitive to the choice of regression method. Mutagen density (IMF×106 per m3 of air) is obtained by multiplying mutagenic potency by ambient OC concentrations. A sample was considered mutagenic if the mean mutant fraction exceeded both concurrent and historical negative controls at the 95% confidence level (CL) at least at one dose level (26) 684

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and the mutagenic potency slope was significantly greater than zero at the 95% CL. The mutagenicity of extracts was compared as described elsewhere (14, 27). Briefly, each set of regression coefficients was compared by conducting an ANOVA to determine whether a difference existed among them. If a significant difference was found, then 95% confidence intervals were constructed, and those samples whose confidence intervals did not overlap were considered different at the 95% CL. Significant differences are designated by different letters (a, b, c), while samples that were not significantly different from each other were assigned the same letters, for example, samples marked ab were not significantly different from either a or b but were different from c. Sensitivity to outliers and the assumption of linearity were tested as described previously (14).

Results and Discussion Mutagenicity of Whole Samples and Their Fractions. The concentration-response data for the unfractionated and

FIGURE 3. Mutagenic potency (IMF×106 per µg of EOC) of extract fractions from PM2.5 collected during 1995 at Brockport (BR) and Rochester (RO) in New York and at Quabbin (QB), Reading (RD), and Kenmore Square, Boston (KS) in Massachusetts. Samples are unfractionated (U), nonpolar 1 (NP1), nonpolar 2 (NP2), semipolar (SP), polar (P), and reconstituted (R). The height of each bar is equal to the slope of the linear fit; error bars represent 95% confidence intervals; different letters (a, b, c) indicate a significant difference at this 95% confidence level (see Experimental Section). a is different from b; ab is not different from a or b but is different from c.

TABLE 1. Distribution of Mutagenic Potency among Fractionsa extract

BR

RO

QB

RD

KS

RD WS KS WS

unfractionated 13% 22% 48% 57% 38% 121% nonpolar fraction 1 0% 2% 6% 2% 10% 10% nonpolar fraction 2 4% 17% 27% 34% 28% 32% semipolar fraction 82% 68% 35% 47% 44% 41% polar fraction 14% 14% 32% 17% 18% 16% reconstituted 77% 60%

176% 1% 41% 41% 18%

a Total mutagenic potency of a sample is equal to the sum of the mutagenic potencies of the four fractions (NP1, NP2, SP, and P). The relative mutagenicity of an extract was found by dividing its mutagenic potency by the total mutagenicity of that sample. RD WS and KS WS are the wintertime samples collected during December 1994 at Reading and Kenmore Square, respectively.

fractionated annual composite samples are presented in Figure 2, and the mutagenic potency (IMF×106 per µg of EOC) of the samples and fractions at each of the five sites are shown in Figure 3. The sum of the mutagenic potencies of the four fractions (NP1 + NP2 + SP + P) from each site was taken to represent the total mutagenic potency of that sample. Table 1 presents the percentage of the total mutagenic potency of the sample contained in each fraction (i.e., the mutagenic potency of the fraction divided by the total mutagenic potency of the sample). The unfractionated extracts accounted for 13-22% of the total mutagenic potency at the NY sites and for 38-57% at the MA sites. Mutagenic potency at each site was unequally distributed among the fractions, and at all of the sites the SP fraction was the most mutagenic, by a factor of 4-5 at the NY sites and by 10-50% at the MA sites. At QB, RO, RD, and KS the NP2 and P fractions were equally mutagenic, while at BR the P fraction was somewhat

more mutagenic than the NP2 fraction. None of the NP1 fractions were mutagenic. Generally, the sum of the mutagenicity of the fractions significantly exceeded that of the unfractionated extracts. An increase in mutagenic potency after fractionation has been observed in other studies (e.g., ref 19) and is possibly explained by reduced interference (e.g., reduced competition for metabolizing enzymes) among chemicals in the fractions. The results are consistent with two trends observed in previous studies of the mutagenicity of airborne particles, evaluated using a variety of fractionation methods and bioassays (11, 28-41): (1) the mutagenicity of airborne particles is highest in urban and industrial centers, and (2) although fractions containing PAH and nitro-PAH are associated with mutagenicity, fractions containing more polar compounds, such as oxygen-containing PAH derivatives, are often the most mutagenic (30, 33, 36, 38, 39). In eastern North America, PM10 samples from Hamilton, Ontario (35), a city ∼200 km west of Rochester and Brockport, were fractionated and tested in bacterial assays. Fractions containing PAH, nitro-PAH, and PAH ketones and quinones contained 84% of the total mutagenicity, similar to our observations that SP fractions contained 85-86% of the mutagenicity at RO and BR and 62-81% at the MA sites. PAH of molecular weight ≈ 252 amu, nitro-PAH, and more polar compounds were found in the most mutagenic fractions, which is also consistent with our results. Nishioka et al. (30) and Lewtas et al. (33) reported on the bacterial mutagenicity of SRM 1649 (total suspended particles collected in Washington, DC). Significant mutagenicity was associated with the acid fraction and with fractions containing PAH and nitro-PAH, although the most mutagenicity was present in fractions containing more polar compounds. VOL. 38, NO. 3, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Mutagen density (IMF×106 per m3 of air) of extract fractions from PM2.5 collected in the northeastern United States during 1995. Error bars represent 95% confidence intervals; different letters indicate a significant difference at this 95% confidence level (see Figure 3). Abbreviations are the same as Figure 3. The annual average organic carbon concentrations (µg OC/m3) for the five sites (expressed as the mean ( standard deviation) are BR 2.72 ( 0.03, RO 3.41 ( 0.04, QB 2.72 (0.04, RD 3.94 ( 0.05, and KS 5.68 ( 0.06 (14). Casellas et al. (36) reported that PAH and nitro-PAH had an important contribution to bacterial mutagenicity in aerosols from Barcelona, Spain, and also identified oxy-PAH in the fractions with the most +S9 activity. Cerna et al. (38) collected PM10 samples from rural and industrial locations in the Czech Republic and found that the slightly polar (containing nitroPAH) and moderately polar fractions were more mutagenic in bacterial assays than the aromatic (PAH) fraction. A few studies of fractionated ambient aerosol have been conducted using assays based on nonbacterial cells. For example, Tuominen et al. tested fractionated particulate samples using sister chromatid exchange in rodent cells and reported that the genotoxicity was mainly associated with the most polar fractions (39). Two recent studies in the Czech Republic tested the same fractions as Cerna et al. (38). Assays based on acellular DNA adducts and on chick embryotoxicity were most affected by PAH and their methyl derivatives (40), while DNA adducts in rat and hamster cells showed the highest genotoxic activity in the fractions containing PAH and nitro-PAH (41). The h1A1v2 assay was used in two previous studies of urban aerosols (19, 20). Both studies demonstrated significant human-cell mutagenicity associated with nonpolar PAH and higher mutagenicity associated with more polar compounds. Durant et al. (22) reported that the two fractions of SRM 1649 containing PAH accounted for ∼20% of the mutagenicity of the entire extract. One of those fractions also contained semipolar compounds, including PAH ketones and quinones. A third fraction containing different classes of oxy-PAH accounted for ∼50% of the total mutagenicity of the extract, and a fraction containing polar compounds accounted for an additional ∼30%. Hannigan et al. (19) fractionated a single annual composite sample combining PM2.5 collected from five sites in the Los Angeles urban center. The mutagenic 686

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potency of the LA unfractionated extract was 0.15 IMF×106/ µg EOC (SD ) 0.03), compared with 0.04-0.1 IMF×106/µg EOC for our five sites. Hannigan et al. found that of the total h1A1v2 mutagenic potency, 46% was present in two fractions containing unsubstituted PAH (compared with 4-19% in NY and 33-38% in MA) and 54% in two fractions containing semipolar and polar compounds, e.g., nitro- and oxy-PAH (compared with 82-96% in NY and 62-67% in MA). Despite some differences in samples and methods, the human-cell mutagenicity associated with the compound classes in each fraction was consistent among Hannigan et al. (19), Durant et al. (22), and the present study. However, the human-cell mutagenic potency of the unfractionated PM2.5 samples was greater in LA than in the northeastern United States. On the other hand, the SP and P fractions contained a higher fraction of the total mutagenic potency in the northeast than in LA. This difference suggests the importance of semipolar and polar mutagens in the northeast samples. It is possible that these more polar mutagens may be produced in primary emissions along with PM2.5; it is also possible that they result from photochemical oxidation during atmospheric transport. Spatial Trends in the Mutagenicity of the Fractions. Mutagenic Potency. We observed interstate differences in the mutagenic potency of some of the fractions, but no significant or consistent intrastate spatial trends (Figure 3f-j). Interstate differences were most pronounced among the SP fractions (Figure 3i), for which the mutagenic potency was ∼2-6-fold higher at the NY sites (0.22-0.27 IMF×106/µg EOC) than at the MA sites (0.04-0.11 IMF×106/µg EOC). Interstate differences were also evident in the distribution of mutagenicity among the fractions: at the NY sites the SP fraction was 4-5 times more mutagenic than the other fractions, accounting for 68-82% of total sample mutagenicity, while at the MA

FIGURE 5. Mutation assay results for fractionated extracts collected at Reading and Kenmore Square during Nov-Dec, 1994 (n ) 20). (a) Dose/response plots: IMF×106 per µg of EOC vs µg EOC per 12 mL assay (see Figure 2 for details). An asterisk (*) indicates doses greater than 600 µg EOC were tested (additional doses of 920 and 856 µg EOC per 12 mL for RD and KS, respectively). (1) Indicates results are sensitive to assumption of linearity (see Experimental Section). (b) Mutagenic potency and (c) mutagen density (see Figures 3 and 4 for details). sites the SP fraction was only 10-50% more mutagenic than the other fractions, accounting for 35-47% of total sample mutagenicity (see Table 1 and Figure 3a-e). Mutagen Density. Mutagen density of the extracts (IMF×106 per m3 of air) is calculated by multiplying the mutagenic potency (Figure 3) by the annual average ambient OC concentration at each site as given in ref 14: BR 2.72 ( 0.03, RO 3.41 ( 0.04, QB 2.72 ( 0.04, RD 3.94 ( 0.05, and KS 5.68 ( 0.06 µg OC/m3. OC concentrations were higher at urban sites, leading to a significant increase in mutagen density with urbanization, especially among the NP2 and SP fractions (Figure 4h and i). In addition, the NP1 and P fractions at KS were significantly more mutagenic than at other sites (Figure 4g and j). Variations in mutagen density were primarily influenced by airborne concentrations of OC rather than by a difference in the mutagenic potency of the OC. This effect was also observed for the bimonthly composites (14).

Few studies have evaluated the spatial variations in genotoxicity of fractionated aerosol extracts. Fractions (acid/ base/neutral) and neutral subfractions (by size and polarity) of PM10 from two Czech cities, Prachatice (a residential area) and Teplice (a industrial/mining 180 km to the north of Prachatice), were tested in bacterial and mammalian cell genotoxicity assays (38, 40, 41). The genotoxicity (per mass of extract) of unfractionated extracts at Teplice was 2-3-fold higher than at Prachatice. In contrast, we observed no significant spatial differences in the mutagenic potency of unfractionated extracts of PM2.5. In the Czech studies the relative genotoxicity among fractions and subfractions showed no spatial differences for rodent cell DNA adducts (41) and acellular DNA adducts (40). However, some spatial differences were observed in the relative genotoxicity of the neutral subfractions. At Teplice, the aromatic subfraction accounted for ∼84% of the total chick embryotoxicity of the VOL. 38, NO. 3, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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neutral fraction, compared with ∼72% at Prachatice (40). The +S9 bacterial mutagenic potency (38) at Teplice was comparable for the slightly and moderately polar subfractions (each containing 30-50% of the mutagenic potency of the unfractionated extracts), while at Prachatice the moderately polar subfraction (40-50%) accounted for more mutagenic potency than the slightly polar subfraction (10-9%). Thus, a greater portion of the genotoxicity was associated with more polar compounds at the residential site, while less polar compounds contributed more to the genotoxicity at the industrial site. Although there were significant differences in genotoxicity and the relative genotoxicity of the subfractions over a distance of close to 200 km between Teplice and Prachatice, it is unclear whether these variations are due to local effects, such as the level of development, or regional effects, such as the district in which each city is located. We observed in the northeastern United States that the mutagenic potency did not vary with increasing urbanization (i.e., within NY and MA over distances of ∼20-100 km); however, significant differences were observed over distances on the order of ∼500 km (i.e., in NY as compared to MA). These findings demonstrate that in the northeastern United States local effects such as urbanization were less important for mutagenic potency than regional effects. Comparison of Annual, Wintertime, and Bimonthly Composite Samples. The mutagenic potency results were sensitive to the choice of how samples were composited. There were no significant differences among our sample sites in the mutagenic potency of the unfractionated annual composites. In contrast, we reported previously (14) that the annual average mutagenic potency of bimonthly composites was ∼2-fold higher at the NY sites than at the MA sites (0.160.19 IMF×106/µg EOC in NY vs 0.07-0.08 in MA averaged over the six bimonthly composites). The average mutagenic potency of the bimonthly composites was ∼3-fold greater than the mutagenic potency of the annual composites at the NY sites, whereas at the MA sites the average of the mutagenic potency of the bimonthly composites was approximately equal to the mutagenic potency of the annual composites. A significant increase in mutagen density was observed, however, along the gradient of increasing urbanization from QB to KS, mainly because of the increase in ambient OC concentrations with urbanization; this trend was consistent with the results of the bimonthly composites. The mutagen density analysis likely showed less sensitivity to the method of compositing than did mutagenic potency because results are dominated by the large variations in the ambient concentrations of OC. These comparisons demonstrate that the interpretation of mutagenicity results for the same samples can differ depending on how the samples are composited: in the present study, only when samples were separated into bimonthly composites did we observe significant spatial differences in mutagenic potency. Our results also showed significant differences between the mutagenicity of unfractionated samples and the sum of their fractions. Most notably, at the two NY sites, the mutagenicity of the semipolar fractions was higher than the unfractionated samples, and the mutagenicity of the semipolar fractions in NY were much higher than in MA. Comparing the mutagenic potency of the wintertime samples at RD and KS (Figure 5) with the mutagenic potency of annual composites (Figure 2), we observed that the unfractionated wintertime extracts (0.15 and 0.13 IMF×106/ µg EOC at RD and KS, respectively) were more mutagenic than the unfractionated annual composites (0.077 and 0.097 IMF×106/µg EOC at RD and KS, respectively). This is consistent with our previous finding (14) that the mutagenic 688

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potency of bimonthly composites collected during the winter was significantly higher than in the summer. Significance of Trends. The results of the present study build upon the findings of previous human cell mutagenicity studies (19, 22) and show that the way in which samples are composited can affect mutagenicity. Thus, to understand variations in mutagenicity, it is necessary to test unfractionated extracts as well as samples separated into chemical fractions and/or seasonal composites. In addition, we found that the relative contribution to mutagenic potency of each chemical fraction varied greatly from one region to another but was not observed to vary with urbanization within regions. Our results also show that the regional differences in mutagenic potency were linked to differences in the mutagenicity of the semipolar fractions, while the other fractions (and unfractionated extracts) exhibited no such variations. Not only were the SP fractions the most mutagenic at all sites, but more significantly, they were mutagenic to different degrees in each region, both in absolute magnitude and relative to the unfractionated extracts and the other fractions. It is possible that either the sources of PM2.5 at the NY sites were enriched in mutagenic semipolar compounds or significant regional differences exist in the rates of chemical transformations during atmospheric transport.

Acknowledgments We would like to take this opportunity to acknowledge the many contributions of Dr. Glen Cass. Glen dedicated his professional life to the study and practice of air pollution research, policy, and education. His expertise included ambient air quality and air pollutant source emissions, design of regional air pollution control strategies, health effects of air pollutants, environmental economics, and policy analysis. Glen died in August of 2001, leaving behind a legacy of students and colleagues who share his passion for air pollution research and education. His enthusiasm and leadership are greatly missed. Lawrence Donhoffner and Lita Doza-Corpus performed the human-cell assays, and Koli Taghizadeh and Elaine Plummer provided support with analytical techniques. Funding for this study was provided by the National Institute for Environmental Health Sciences: Mutagenic Effects of Air Toxicants grant (P01-ESO7168), Superfund Hazardous Substances Basic Research grant (SF P42-ESO4675), and MIT CEHS Core grant (P30-ESO2109).

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Received for review July 7, 2003. Revised manuscript received October 28, 2003. Accepted November 11, 2003. ES0347282

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